Hemophilia A (HA) is a common disorder of coagulation caused by deficiency of factor VIII (FVIII). The mainstay of therapy has been replacement therapy with recombinant human FVIII. However, because of high cost, access to therapy, and inhibitory antibody formation, patients continue to suffer from significant long-term morbidity and mortality. The generation of improved versions of FVIII molecules with increased specific activity and/or improved immunological profile would improve hemophilia treatment. The expression of these new variants with liver-directed gene therapy in conjunction with strategies to tolerize patients to the therapeutic protein would constitute important clinical advances. Our development of an optimized helper-dependent adenoviral gene vector (HDV) system has enabled us to achieve long term expression of both secreted and intracellular transgenes with absent chronic toxcity and persistence of vector in small and large animal models. However, acute toxicity remains an obstacle to clinical translation. To overcome this, we have developed a novel balloon catheter based delivery system into the hepatic circulation. We have also developed a novel method for generation of tolerogenic dendritic cells based on calcium phosphate-dependent transduction of HDV expressing TGF2 and IL10. Based on these preliminary data, we will address three important questions in genetic therapy for hemophilia a) Can we increase the therapeutic index by maximizing gene expression of more bioactive FVIII variants? b) Can we increase the maximal toxic dose by overcoming the threshold/nonlinear dose response of Ad gene transfer in canine HA and achieve re-administration of the vector? c) Can we tolerize the recipient of gene transfer or h FVIII by adoptive transfer of autologous tolerogenic DCs? The overall goal of these studies is to address the major obstacle to clinical translation of adenoviral gene therapy in HA: 1) To achieve clinical correction in a large animal model of HA at a dose relevant for human translation and 2) to develop an antigen-specific tolerizing strategy for FVIII expression after gene transfer.